1. Field of the Invention
The present invention relates to selectively labeled molecules and the use of nuclear magnetic resonance spectroscopy to detect and characterize molecular interactions between the selectively labeled molecules and putative ligands.
2. Background of the Invention
Random screening of synthetic and natural product libraries to discover compounds that bind to a particular target molecule is a common method for obtaining new pharmaceutical entities. A target molecule is typically exposed to one or more compounds and assays are performed to determine if complexes between the target molecule and one or more of the compounds are formed. Once random screening has identified a potential candidate, or lead compound, analogs of the lead compound are synthesized in an effort to improve binding and selectivity. The analogs are then screened, usually by the same assays used to identify the lead compound.
A common problem associated with current screening methods involves non-specific ligand binding; that is, the ligand attaches to the target molecule but in a non-specific manner. Such non-specific binding often occurs and is difficult to detect. In such cases, optimization of ligand binding is greatly complicated. The full potential for developing pharmaceutical-based therapies depends not only on screening vast numbers of compounds against a target, but also on determining specific details concerning interactions between the ligand and the target molecule.
Nuclear magnetic resonance (NMR) spectroscopy is known for its ability to characterize macromolecular structures, and is a technique for investigating both static and transient features of ligand binding to a target molecule (Pellecchia, et al., Nature Rev Drug Disc 2002, 1:211). NMR spectroscopy is a useful tool for determining the binding of ligands to target molecules, and has the advantage of being able to detect and quantify interactions with high sensitivity without requiring prior knowledge of protein function. Furthermore, NMR spectroscopy can provide structural information on both the target and the ligand to aid subsequent optimization of weak-binding hits into high-affinity leads.
Methods of detecting binding of a ligand compound to a target biomolecule by generating first and second nuclear magnetic resonance correlation spectra from target biomolecules which have been uniformly labeled are reported in U.S. Pat. Nos. 5,698,401 and 5,804,390. The first spectrum is generated from data collected on the target substance in the absence of ligands, and the second in the presence of one or more ligands. A comparison of the two spectra permits determination of which compounds in the mixture of putative ligands bind(s) to the target biomolecule.
Despite the broad applicability of using NMR spectroscopy to determine molecular interactions, its use with macromolecules is complicated by the extremely complex spectra associated with macromolecules. Although, isotope labeling in macromolecular NMR spectroscopy can result in increased sensitivity and resolution and in reduced complexity of the NMR spectra, attempts to improve NMR analysis of macromolecules by labeling target molecules with NMR active isotopes has been only marginally successful. Isotope labeling would promote the efficient use of heteronuclear multi-dimensional NMR experiments and provided alternative approaches to the spectral assignment process and additional structural constraints from spin-spin coupling.
Current methods of assessing molecular interaction that utilize NMR spectroscopy typically rely on uniform labeling of the target molecule. When using uniform labeled target molecules, a complete 1H and 15N resonance assignment of at least the backbone nuclei of the target molecule must be completed before any ligand-protein interactions can be assessed by NMR spectroscopy. The assignments are then used to map the interactions of a ligand by following chemical shift changes upon complexation. This process can be quite lengthy and is generally limited to small-to-medium-sized proteins due to line broadening and spectral overlap with larger proteins. Even with the advent of TROSY (Transverse Relaxation Optimized SpectroscopY) (Pervushin, et al., Proc Natl Aca. Sci USA 1997, 94:12366; Wüthrich, Nat Struct Biol 1998, 5:492) and perdeuteration, the problem of complete resonance assignments in target molecules remains a major stumbling block for NMR ligand-binding studies.
There have been attempts to overcome the problems associated with uniform labeling of target molecules by attempting to selectively label the methyl groups of the amino acids isoleucine, methionine, threonine, alanine, leucine, and valine. However, these amino acids often are present in the interior of the protein and are not involved in ligand or protein binding.
Thus, there is a need for methods for determining molecular interactions by NMR spectroscopy that result in a simplification of the spectrum and enable the study of significantly larger macromolecules. Also needed are improved methods for high-throughput screening using NMR spectroscopy.